Methods and systems for sampling, screening, and diagnosis

ABSTRACT

Apparatus, systems, and methods for detecting, screening and sampling of cells are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/541,781, filed Feb. 4, 2004; 60/577,790, filed Jun. 8, 2004; and60/619,621, filed Oct. 18, 2004. The contents of each of theseapplications is hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

Biological monitoring of the growth of cells has applications in fieldssuch as microbiology and medicine. Methods for detecting the growth ofmicroorganisms are well-developed in microbiology laboratories; however,such techniques cannot always be easily applied to real-worldsituations. For example, isolation and identification of an unknownmicroorganism can be difficult and time-consuming, particularly when themicroorganism is not suited to growth under typical laboratoryconditions.

In some cases it is important to study a microorganism in its natural oradopted environment. For example, bioremediation is a recent approach todecontamination of polluted land, water and marine sites, in whichmicroorganisms can be used to remove or destroy contaminants in situ.While bioremediation has been demonstrated to work in some cases, it isoften difficult to predict which microorganisms will be most useful at agiven site, or which conditions will most effectively promote thebioremediation process. Without considerable information about the typesof pollutants and indigenous microbes present at a contaminated site,the selection of appropriate bioremediation conditions is difficult.Unfortunately, due to the relatively inaccessible nature of somecontaminated sites (for example, underground water), obtaining suchinformation can be difficult. Conventional sampling and testing can belabor-intensive and time consuming.

The inability to easily and rapidly obtain accurate sampling informationalso hinders efforts to discover novel microorganisms or naturalproducts, a process often referred to as bioprospecting. Bioprospectingfrequently involves searching for organisms in inhospitable orinaccessible environments, such as hot springs, black smokers, deepocean, and other locales having extreme physical or chemical conditions.However, without adequate means for obtaining complete information aboutthe prevailing conditions and microbial communities in these areas,bioprospecting efforts can be slow and difficult.

While many approaches to these problems have been proposed, few methodshave been developed which are rapid, inexpensive, and easily customizedfor the study of a wide variety of microorganism types and environments.

U.S. Pat. No. 6,187,530 discloses an aquatic autosampler having multiplefilters; the device can serially gather samples of microorganisms byexposing one filter at a time to a water sample. However, the devicecannot simultaneously collect multiple samples.

PCT Patent Publication WO 2004/081530 discloses an in situ microcosmarray (ISMA) technology suitable for environmental monitoring andbioprospecting. The contents of that application are hereby incorporatedby reference in their entirety.

Once a sample for study has been obtained, there is a need for rapid andsensitive identification of compounds and microorganisms present in thesample. While many approaches to this problem have been suggested, fewmethods are capable of detecting species of interest without extensivesample clean-up and purification steps, which can be time-consuming andexpensive.

SUMMARY OF THE INVENTION

The present invention provides improved devices, systems, and methodsfor monitoring environments, e.g., for use in bioremediation,bioprospecting or medical testing and screening. The devices of theinvention can be simply made, can be reusable or disposable, and can beused without contaminating the environment being monitored.

The devices can also be made to be compatible with standard automatedsample handling systems, thereby permitting automated, high-throughputanalysis of samples obtained from the environment under study. Analysisof microorganism-containing samples can be performed using massspectrometric methods, which can provide rapid, accurate determinationof microbial species and/or determine the levels or types of proteins orother cell products produced in a given environment.

The inventive device is useful for monitoring sensitive environments inwhich contamination is to be avoided. In certain embodiment, the deviceincludes an effluent collection reservoir, for collecting the effluentfrom the capillary compartments or reagent reservoirs. In this way,release of materials from the interior of the device (fluids, biologicalorganisms or cells, chemical compounds, test compounds, and the like)can be avoided. Thus, in one embodiment, the invention provides animproved, lower cost method for environmental monitoring andbioprospecting.

In another embodiment, the invention provides an improved bioremediationassessment method and tool.

In another embodiment, the invention provides an improved bioremediationassessment method and tool that will enable the automated, large-volume,high-throughput analysis of bioremediation sites.

In another embodiment, the invention provides a monitoring method, tooland analysis strategy that allow for the automated, rapid andsimultaneous determination of the following parameters: (1) fluidquality and toxicity, (2) intrinsic bioremediation potential, (3)accelerated bioremediation potential following nutrient amendment, (4)effective bioaugmentation strategies for environmental cleanup, (5)turnover rates of natural compounds and environmental pollutants undernatural and enhanced conditions, (6) in situ DNA synthesis and proteinexpression, (7) in situ growth/death rates and metabolic activity ofnative and introduced biological agents under natural and alteredenvironmental conditions, (8) structure and dynamics of microbialcommunities indigenous to natural environments, and (9) identity andactivity of microorganisms of potential value for use in biotechnology.

In another embodiment, the invention provides a monitoring method andtool that may be applied to assess the potential risk resulting from therelease of chemicals, potentially hazardous materials, non-indigenousmicroorganisms, pathogens and genetically engineered microorganisms intonatural environments.

In one aspect, the invention provides a method for determining thepresence, absence type, or amount of microbes in an environment, withoutcontamination or perturbation of the environment.

In another aspect, the invention provides a method for rapidlydetermining the optimal growth conditions for a cell in an environment.

In another aspect, the invention provides a method for simultaneouslytesting a variety of agents to determine the type and amount of an agentcapable of inhibiting or stimulating cell growth.

In another aspect, the invention provides a method for screening ofvarious conditions to optimize therapeutic treatments.

In another aspect, the invention provides a method for detectingparasites.

In another aspect, the invention provides a device for measuring thegrowth of cells in an environment.

In another aspect, the invention provides a device for identifying acell present in or isolated from an environment.

In another aspect, the invention provides a system for characterizingmicroorganisms or test compounds present in an environment.

These and other features and embodiments of the invention will be betterunderstood by reference to the description and claims below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a scheme showing one embodiment of a system according to theinvention.

FIG. 2 is a schematic representation of a preferred embodiment of adevice according to the present invention.

FIG. 3 is a cross-sectional view of the housing shown in FIG. 1, withenlarged representations of a valve plate adjacent to capillary inletsin both their open and closed positions.

FIG. 4 is an exploded view showing a valve plate of FIG. 1 and thecomponents that are used to cause it to move laterally to open and closethe capillary's inlet.

FIG. 5 is a schematic representation of a preferred embodiment of thepresent invention being extended down a groundwater monitoring well.

FIG. 6 is a scheme showing a method for identifying biological cells,including microorganisms such as bacteria.

FIG. 7 is a scheme showing a method for identifying biological cells,including parasites.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides systems, devices, and methods for monitoring testenvironments, including biological fluids, preferably withoutcontamination of the test environment.

In one aspect, the invention provides a device for sampling or studyinga test (e.g., environmental) fluid. The device includes a) a housinghaving at least one opening in a wall thereof; b) an array assemblydisposed within the housing and comprising a plurality of capillarymicrocosms, each capillary microcosm having a fluid inlet, a fluidoutlet, and a capillary chamber; c) a fluid manifold in fluidcommunication with the opening in the housing and the plurality ofcapillary microcosms; and optionally d) an effluent reservoir forcollecting effluent from the fluid outlets of the capillary microcosms.

In preferred embodiments, the device includes at least one effluentreservoir or container in fluid communication with a fluid outlet of atleast one capillary compartment or chamber, for collecting and retainingthe effluent from the chamber(s). The effluent reservoir or containermay take the form of a rigid container, a flexible bladder, or abibulous or absorbent material for absorbing fluid. The effluentreservoir can be situated within the device housing, or it can besecured through a fluid-tight seal to a fluid outlet of the devicehousing. The effluent reservoir or container preferably is locatedwithin the device housing. By retaining the effluent within the housing,the possibility of fluid leakage (and resultant contamination of theexternal environment) can be minimized.

By collecting the effluent from the capillary compartments, materialsfrom the interior of the device (biological organisms or cells, chemicalcompounds, test compounds, and the like) are not released into theenvironment external to the device. Thus, the devices and methods of theinvention are well-suited for use in sensitive environments which shouldnot be perturbed.

Examples of sensitive environments include: in vivo uses, where thedevice is implanted into a human or animal body; surface or subsurfacewater sources, including surface reservoirs and sub-surface aquifers orwater tables; food supplies; sterile environments (such as laboratoriesor hospital operating theaters); clean rooms for manufacturing;environments in which introduced organisms could be harmful (including,e.g., probes for use in outer space and planetary exploration); and thelike. In certain applications, a sensitive environment can be tested byplacing the device of the invention outside an environment of interestbut is in fluid communication with the environment. For example, fluidscan be piped or flowed from an environment of interest to the device ofthe invention for testing.

In a preferred embodiment, the test environment contains a fluid, suchas water, an aqueous solution, or an aqueous bodily fluid (such asblood, serum, plasma, saliva, urine, cerebrospinal fluid, or the like),in which the device of the invention is immersed. In another embodiment,the test fluid is a gas, e.g., an atmospheric gas such as air.

In a preferred embodiment, there is a separate effluent reservoirassociated with each capillary chamber or microcosm within the array. Bycollecting effluent from each chamber individually, the effluent of eachmicrocosm can be saved for later analysis when the device is removedfrom the test environment, permitting data to be obtained for eachindividual microcosm.

In another embodiment, the invention provides a method for optimizingmedical treatment for a patient either in vivo or ex vivo (e.g., invitro). The method includes the following steps: a) providing a devicecomprising a housing and an array of capillary microcosms, each of thecapillary microcosms being in fluid communication with a fluid manifold,and each of the capillary chambers containing either i) a first sampletype associated with a disease state of the patient, ii) a second sampletype not associated with the disease condition, or iii) both a firstsample type and a second sample type; b) subjecting each of said samplesto a treatment condition; c) determining the effect of the treatmentcondition on each sample; and d) selecting an optimized medicaltreatment for the patient. It will be appreciated that the first andsecond sample types can be, e.g., samples of diseased and normaltissues, respectively; samples of potential donor and recipient tissue,e.g., for evaluating the feasibility of transplants, and the like.

In another embodiment, the invention provides a method for optimizingmedical treatment for a target population (e.g., a particular species,or a subpopulation of a larger population). The method includes thefollowing steps: a) providing a device comprising a housing and an arrayof capillary microcosms, each of the capillary microcosms being in fluidcommunication with a fluid manifold, and each of the capillary chamberscontaining either i) a case sample associated with a disease stateoccurring in the target population, ii) a control sample or iii) both acase sample and a control sample; b) subjecting each of said case andcontrol samples to a treatment condition; c) determining the effect ofthe treatment condition on each sample; and d) selecting an optimizedmedical treatment for the patient. In this embodiment, the controlsample can be, e.g., a sample from a member of the larger population whois unaffected by a particular condition.

The Housing

The material for the exterior of the housing is preferably selected foruse in the test environment. For example, the housing can be rigid foruse in demanding applications, or flexible, e.g., for use where thedevice is to be worn on or even in (e.g., implanted in) a living body.To avoid contamination, in certain embodiments the exterior of thehousing can be washed, decontaminated, disinfected, and/or sterilized,e.g., by heating, by irradiation, use of chemical sterilants such asalcohol, and the like. A suitable material will be selected to becompatible with such conditions. The material for the housing can alsobe selected to resist extremes of temperature, pressure, pH, corrosion,or other adverse conditions that may be present at the selected testsite, either by using an appropriate body wall material or by coating abody wall material with an appropriate surface chemistry.

In certain preferred embodiments, the housing is constructed ofmaterials such as steel, aluminum, or plastic, although other materialscan be selected. Titanium or other biocompatible materials can be usedfor devices intended for in vivo use.

The housing will generally be selected to be of a size and shape adaptedfor the intended use of the device, e.g., sized to fit within a boreholeof a well, or miniaturized for implantation into a living body. Inaddition to containing the capillary array, the housing may containother components, such as the fluid manifold, a battery or other powersource (e.g., a fuel cell), an effluent reservoir, pumps, valves,reagent reservoirs, sensors, signal transduction equipment, and thelike.

In certain preferred embodiments, microprocessor control circuitry,e.g., for the control of valves, pumping devices, controls, sensors andother electronic controls, or for acquisition of data from such devices,are integrated into the device to form an integrated unit (e.g., seebelow).

The Capillary Array

The capillary array includes a plurality of test chambers, ormicrocosms, each adapted for containing a cell sample (e.g.,microorganisms or tissue samples) or a sample of a test compound. Eachchamber has a fluid inlet and a fluid outlet; both the inlet and theoutlet can be controlled with valves to control the flow of fluid intoand out from each chamber. Such controls can be configured to permitcontrol of fluid flow through the chambers individually, or to permitoperation of certain chambers simultaneously. A check valve can beprovided at the outlet of each chamber to prevent backflow of fluid intothe chamber.

In one embodiment, the capillary array contains a standardized number ofcapillary chambers (e.g., 96, 384, 1536) corresponding in size andconfiguration to standard microtiter plates. By using a standard sizeand footprint for the capillary array, it is possible to usestandardized robotic handling equipment (e.g., liquid handlers) to load,unload, or analyze the contents of the chambers. In one embodiment, thecapillary array comprises a block of polytetrafluoroethylene (Teflon)resin. Customized microtiter formats include plates having a pluralityof capillary chambers (e.g., 48, 100, or 100,000 capillary chambers.

The capillary chambers or compartments (microcosms) of the device can beprovided with a variety of materials for adjusting or perturbing theconditions within the chamber. For example, a chamber may optionallyinclude a substrate (such as a filter or other material, e.g., glasswool) suitable for collecting bacteria from a fluid flowing through thechamber. Filter materials can be selected from manycommercially-available sources, for example, nitrocellulose filters,nylon membrane filters, and the like. In certain embodiments, a filtermaterial is compatible with both the collection of microorganisms (orother cells) and subsequent culture of the microorganisms. In additionto filtration, microorganisms, compounds or particles can be collectedin the microcosms by sorption, precipitation, sedimentation,coagulation, extraction, chromatography, affinity separation, sizeexclusion separation, passive attachment to presented surfaces, oractive attachment to presented surfaces.

The capillary array can be used for capturing, isolating, growing, ortesting a variety of cell types, including bacteria, fungi (includingyeasts), parasites, protozoa, and cells or tissue samples (such asbiopsies) isolated or obtained from multi-cellular organisms (e.g.,tissue culture of plant cells, animal cells, insect cells, mammaliancells, and the like). The chambers can also be used for the study ofviruses, prions, and other infectious particles. Thus, the systems andmethods of the invention can be used to study viruses, prokaryotesincluding, but not limited to, bacteria, and Archaea, and eukaryotesincluding, but not limited to, yeasts, fungi, protozoa, plant cells,animal cells, and mammalian cells. The term “microorganism,” as usedherein, is intended to include bacteria, fungi, parasites, protozoa, andviruses, and further includes spores, seeds, and other vegetative formsof microorganisms. The term “cell”, as used herein, is intended toencompass living cells, whether of single-celled or multi-cellularorganisms, and also include spores, seeds, larvae, and other forms.

A chamber can include a growth medium or food source for growth of cellor tissue cultures. In certain preferred embodiments, the food sourcecan be specific to a particular cell type (such as a specificmicroorganism) which is to be studied. For example, as describedelsewhere herein, Sphingomonas wittichii Strain RW1 is a bacteriumcapable of degrading dioxins and dibenzofurans. Few other bacteria arecapable of using dibenzofuran as a food source; thus, a test chambercontaining dibenzofuran can be used to selectively culture S. wittichiiStrain RW1. As described elsewhere herein, such a food source can beisotopically labeled to provide additional information about any cellscapable of growth, metabolism, reproduction, and production ofbiomolecules and parasites (e.g., viruses) in the presence of the foodsource. As an alternative, a chamber can be loaded with one or moremicroorganisms that were grown on isotopically labeled food sources andwhose growth, metabolism, reproduction, and production of biomoleculesand parasites (e.g., viruses) can be tested by determining the loss ofisotopes or redistribution of isotopes over time.

A chamber can also include test compounds for determining whether a cellcan grow in the presence of the test compound, e.g., the test compoundcan be an antibiotic, an antineoplastic agent, and the like. Thesecompounds can be provided in a matrix or formulation that allows gradualrelease of the compound into the chamber.

The chambers can also be provided with means for containing cells,reagents, or other materials within the chamber. For example, a membranecan be used to retain cells or certain compounds while allowing othermaterials to pass through the chamber and into an effluent collectionreservoir.

It will be appreciated by the skilled artisan in light of the presentdisclosure that the devices and methods of the invention can be used totest for many properties of substances other than cells. For example,the chambers of the device can be loaded with candidate pharmaceuticals(e.g., to test for desirable activities, as well as undesirable sideeffects); with candidate biocompatible materials (e.g., to test for lowimmunogenicity, resistance to biofilm formation, anti-coagulantproperties, resistance to corrosion, and the like); or with potentialtoxins (e.g., environmental contaminants, biowarfare agents, compoundsfor use in commercial products, e.g., preservatives, plasticizers, andthe like). Such materials can be tested to determine the suitability ofa material for use in an environment (e.g., to determine durability orwear qualities such as resistance to corrosion, dissolution, erosion,hydrolysis, or the like) or to determine the effect the compound has onthe environment (e.g., toxicity, changes to pH, corrosiveness, and thelike).

In addition, reagents can be carried in a reagent reservoir, e.g., areservoir within the device housing, which is in fluid communicationwith the fluid manifold, or directly with a chamber or chambers of thearray. Valves and a pump or pumps control release of a reagent orreagents from the reagent reservoir to a capillary chamber or chambersas needed. Examples of such reagents include compounds undergoingtesting (e.g., as described above), or materials for use theexperimental conditions established in a chamber (e.g., growth media,preservatives, fixatives, dyes, nucleic acid hybridization probes,antibodies, cytotoxic agents, antibiotics, salts, enzymes for cellmanipulation (e.g., trypsin for cell digestion) and the like.

Microscale Devices

Especially when in vivo monitoring is desired, miniaturization of thedevice is advantageous. Thus, in certain embodiments, the device is amicro-electromechanical system (MEMS). MEMS technology generallyinvolves fabrication of microscale devices from silicon wafers (see,e.g., M. J. Madou, Fundamentals of Microfabrication: The Science ofMiniaturization, 2^(nd) ed. (CRC Press, 2002)). For example, in oneembodiment, a capillary chamber is etched into a silicon substrate;microchannel fluid pathways, valves, and pumping devices for pumpingfluid to the capillary chambers are similarly made on the siliconsubstrate. The device can also incorporate miniature flow sensors. Inthis embodiment, each fluid flow into each capillary chamber can becontrolled individually and precisely, thereby enhancing thecapabilities of these devices. The flow sensors also allow detection ofblockage or other abnormal flow conditions within the device.

In preferred embodiments, microprocessor control circuitry for thevalves, pumping devices, controls, sensors, and other electroniccomponents of the device are integrated into the MEMS substrate chip toform an integrated device.

Only a very small amount of power is required to operate a MEMS device;the power can be supplied from an on-board battery or via radiofrequency(RF) power supplied by an external RF power source. RF technology iswell suited to implanted devices, which can then be powered, operated,and monitored remotely.

A microscale device can be constructed as described in U.S. Pat. No.6,653,124, the contents of which are incorporated herein by reference intheir entirety. For example, a substrate layer can be etched ormicro-machined with an array of microchambers for containing biologicalcells or chemical compounds to be detected or tested, fluid channels forcommunication with the chambers, and microfluidic devices such as valvesand pumps for directing fluid flow to the chambers.

If biological cells or test compounds are to be provided within themicrochambers prior to emplacement of the device in a test environment,such test materials can be printed or dispensed into the microchambersusing automated slide spotters or microprinters capable of deliveringmicro- or nanoliter-scale spots or droplets of the materials to beincluded in the microchambers.

The substrate layer is then adhered or attached to a covering layer,optionally with a spacer (optionally a membrane for sealing andisolating the microchambers) to provide separation between the twolayers, to provide a miniaturized device according to the invention.

The entire device can be encapsulated within a layer or envelope of asuitable biocompatible material, making provision for an opening foraccess of the external fluid to be tested to the interior of the device.The opening can be or may contain a selective barrier, e.g., a membraneto prevent entry into the device of large particles which could foul orclog the device.

Sensors

A device of the invention optionally include one or more sensors formeasuring a state or property of the cells or fluids within the device.For example, a sensor can be located in (or downstream from) a chambercontaining cells, so that fluid passing over or through the cells isdirected to the sensor for measurement of a property of interest. See,e.g., U.S. Pat. No. 6,806,543 for an example of a microfluidic apparatuscomprising a sensor.

By incorporation of sensors into the device, it is possible to monitorthe conditions in the environment in real time. Such monitoring isuseful for determining, e.g., when a pre-selected end-point for anexperiment is reached (e.g., when a collection reservoir or effluentcontainer is full, when all of an added compound has been consumed bymicrobial growth, when all cells have been killed by a treatment), sothat the device can be removed from the environment and analyzed,reused, or discarded.

Examples of sensors include electrochemical sensors, such as oxygensensors, e.g., for measuring biological oxygen demand (BOD) (see, e.g.,U.S. Pat. No. 6,689,602 for a compact oxygen sensor suitable formeasuring BOD) and glucose sensors, for measuring glucose concentrationof a fluid, e.g., blood or urine (see, e.g., U.S. Pat. Nos. 6,815,186,6,721,587 and 6,673,596); temperature sensors; pressure sensors; opticalsensors, e.g., for performing fluorescence assays of cells within thechambers in real-time; and the like.

A sensor can be wired to transmit information to a control ordata-handling element, such as a microprocessor, which may be anintegral part of the ISMA device or may be external to the device (e.g.,a remote control unit). Wires or leads for communication of a sensorwith a control or data-handling element can be provided according towell-known principles.

Operation of the Device

In operation, the device is first readied by addition of any biologicalcells or materials, or chemical reagents needed, as described above. Thedevice is then placed into the test environment, which, as describedherein, can be a well, a living body, a catheter line or other fluidconduit, or any other environment in which in situ testing is desired.The device can be placed into the test environment remotely, e.g., usinga robotic arm or other mechanical device. To prevent contamination ofthe test environment, the device can be disinfected, washed, orsterilized prior to placement in the environment.

The device can be operatively connected to a control unit for operationof the device. Such a connection can be a wired connection, permittingsignals from the control unit and data from the device to be exchanged,or the device and control unit can be equipped with transceivers forreceiving control signals and transmitting data by radio telemetry.

When the device has been placed in the test environment, a valve (orother device adapted for starting, stopping and/or metering fluid flow)is opened, admitting fluid to the interior of the device and into thefluid manifold. Fluid is admitted into the individual chambers bycontrolled opening and closing of valves (see, e.g. FIG. 2). By exposingvarious chambers to the fluid over a period of time, changes in thefluid composition over time can be observed.

When the device has been placed, real-time data can be obtained fromsensors as described above, to monitor the progression of theexperiment. The sensors can provide information such as, e.g., whetherthe device is functioning properly, when a chamber is full or empty,whether cells are growing (e.g., by monitoring BOD), and the like.

The monitoring or testing process can be allowed to proceed for apre-determined time and then terminated, or else real-time data can beused to determine when the experiment should be ended. For example, anexperiment can be ended when all chambers of the microcosm array havebeen filled with fluid, when all reagents contained in a reagentreservoir have been consumed, when sensor measurements indicate that nofurther bacterial metabolism is occurring, or the like.

When the monitoring or testing process is complete, the device isremoved from the test environment, e.g., by retrieving the device via anumbilical or removing the device from a test fluid sample.

Once the device is removed from the test environment, the identity ofany microbes or cells present in the capillary chambers can bedetermined by a variety of methods, some of which are known in the art.Such methods include genomic, proteomic, physical, chemical, andbiochemical approaches.

Detection and Identification of Microbes and/or Cells

In one embodiment, the invention provides a method for detecting and/oridentifying cells (e.g., microorganisms) having a pre-determinedphenotype. The method comprises the steps of providing a sample fortesting; and detecting by mass spectroscopy the presence or absence of abiomarker diagnostic of, e.g., a microorganism having the pre-determinedphenotype in the sample. A biomarker can be selected prior to beginningthe analysis. The sample can be, e.g., a sample from a device of theinvention, e.g., a sample from a capillary microcosm. The presence oramount of the biomarker in the sample can then be correlated with thepresence of the microorganism having the pre-determined phenotype.

As used herein, the term “biomarker” refers to any detectable biological(e.g., metabolic) product useful as a marker or signal for the presenceof a cell, e.g., a microorganism. A “pre-determined phenotype” refers toa phenotype for which a biomarker is known or can be determined. Abiomarker is diagnostic for, or indicative of, a pre-determinedmicroorganismal phenotype when detection of the biomarker provides areliable indication that a microorganism having the pre-determinedphenotype is present in a sample being tested.

The selection of a suitable biomarker will be routine for one of skillin the art. Preferably, a biomarker will be unique to a pre-selectedphenotype; that is, the biomarker will be specific to the species andphenotype of interest. The biomarker should also be capable of detectionby an analytical system such as a mass spectrometer; for certainembodiments, it is necessary that a biomarker be ionized or ionizablefor mass spectrometric analysis. In preferred embodiments, a biomarkeris a peptide or protein, and the sequence of the peptide or protein isknown; preferably, the sequence is stored in a computer database for usein analytical determination of the biomarker, e.g., by MS.

In contrast to conventional methods of bacterial detection, the methodsof this invention can provide information not only about the presence orabsence of a particular cell type (e.g., bacterial type) or species(e.g., the genotype of a bacterium), but also about the expressedphenotype of the cell (e.g., the types and amounts of gene productsproduced by the cell, e.g., bacterium). In some cases, the phenotype ofthe microorganism depends upon the environment in which the organism isgrown. For example, as described in further detail herein, certainbacteria express dioxin dioxygenase only when grown in the presence of asuitable substrate for the enzyme. The presence of this enzyme in asample therefore demonstrates not only the presence of the particularspecies of bacterium, but also that the bacterium is capable ofproducing the dioxygenase, and that a suitable substrate for the enzymeis present in the bacterial environment. As is described in more detailelsewhere herein, an enzyme or other protein can be detected bydetection of the whole enzyme or by fragments of the enzyme. Thus, aportion of a characteristic enzyme can serve as a biomarker for thepresence of the organism of interest.

Thus, the invention provides methods for determining whether a cell(e.g., a bacterium) having a selected property (e.g., a cell capable ofperforming a pre-selected function) is present in a sample orenvironment. For example, the invention provides methods for determiningwhether a bacterium capable of biodegrading a substrate is present in asample (e.g., by detecting a biomarker diagnostic for suchbiodegradation activity). As another example, the methods of theinvention can be used to study in near-real time the level of functionalenzymes in biotechnology production fermenters and reactors, permittingquality control of processes and products.

As an example, monooxygenase and dioxygenase enzymes are frequentlyassociated with microorganisms (e.g., bacteria or fungi) useful inbioremediation, because such enzymes are capable of degrading a varietyof substrates. In certain embodiments, the bioremediation application isremoval or degradation of aromatic hydrocarbons (e.g., dioxins, toluene,xylene, naphthalene biphenyl, styrene, 2,4,6-trichlorophenol, and thelike). In certain embodiments, the bioremediation application is removalor degradation of nitroalkanes or nitroaromatic compounds (e.g.,2-nitropropane or 2-nitrotoluene), which may be the result of industrialproduction of pesticides, explosives, dyes, pharmaceuticals andplastics. In certain embodiments, a method of the invention comprisesdetecting the presence or absence of a monooxygenase or dioxygenaseenzyme in a sample, with the presence of the enzyme being associatedwith the presence of a bacterial phenotype useful for bioremediation. Incertain embodiments, the enzyme, is an oxygenase, such astoluene-o-monooxygenase, methane monooxygenase, styrene monooxygenase,xylene monooxygenase, squalene monoxygenase, cyclohexanone monoxygenase,butane monoxygenase, 2,4,6-trichlorophenol 4-monooxygenase, and thelike. In certain preferred embodiments, the enzyme is a dioxygenase suchas dioxin dioxygenase, naphthalene dioxygenase, biphenyl dioxygenase,phenanthrene dioxygenase, toluene dioxygenase, 2-nitrotoluenedioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase, catechol1,2-dioxygenase, protocatechuate-3,4-dioxygenase, 2-nitropropanedioxygenase and the like. In a preferred embodiment, the enzyme isdioxin dioxygenase and the microorganism is Sphingomonas wittichiiStrain RW1. In another embodiment, the enzyme is the dioxygenasegi|37727200 of Pseudomonas putida KT2440.

In certain preferred embodiments, biomarker activity or level in amicroorganism of interest (e.g., microorganisms having a pre-determinedphenotype, bacterium capable of performing a pre-selected function) in asample is induced or upregulated in the microorganism prior to analysisof the sample to detect the biomarker. By inducing higher levels ofexpression of the biomarker, detection of the biomarker (and thereforethe microorganism) is improved. Induction of enzymes can be achievedaccording to known methods. For example, as described herein, the amountof dioxin dioxygenase produced by S. wittichii Strain RW1 can bemodulated (i.e., increased or decreased) by selection of appropriategrowth media. Similarly, production of toluene dioxygenase byPseudomonas putida Strain TVA8 can be increased by addition of anappropriate concentration of toluene in the growth medium. By increasingproduction, expression, or levels of a biomarker in a sample, detectionof the biomarker is simplified, and the need for sample clean-up,preparation or other manipulation prior to analysis can be reduced. Asdescribed elsewhere herein, a device of the invention, comprising anarray of microcosms suitable for growth or culture of microorganisms,can be used to investigate the effects of a variety of growth conditionson the production, expression, or level of a biomarker in a sample.

In certain embodiments, samples are subjected to minimal processing orclean-up steps prior to analysis. For example, MS techniques are capableof detecting biomarkers in digests of whole cells (see, e.g., Examples2-5 herein and FIG. 6). By analyzing pre-determined biomarkers that arerelevant for an activity of interest (e.g., bioremediation), onlymicroorganisms relevant to the activity will be detected if present; thepresence of other organisms which do not have the desired phenotype maynot interfere with the analysis. Moreover, as described above, byselecting conditions (e.g., carbon source, temperature, and the like)which favor the expression of relevant biomarkers, greater robustness ofdetection can be achieved, and the need for sample clean-up can bereduced.

The invention also provides methods for determining whether amicroorganism capable of infecting a subject (including a human) ispresent in a sample, e.g., by detecting a protein characteristic of aninfective state of such a microorganism (such as a parasite). In certainembodiments, a method of the invention comprises detecting the presenceor absence of a protein associated with an infectious form or stage of amicroorganism, including a parasite.

One analysis scheme according to the invention is shown in FIG. 6. Asshown in FIG. 6, a bacterial sample (such as a culture, e.g., a cultureobtained from a device of the invention) is harvested or sampled, andwhole cells or sonicated fractions of cells are subjected to enzymaticdigestion with trypsin. Following digestion, optional sample clean-upwith a microcolumn is followed by preparation of the sample for massspectrometric analysis; shown is the preparation of a matrix for MALDIMS. The data obtained from the MS analysis is then analyzed and comparedto a database for identification of peptides or peptide fragmentspresent in the sample, which indicate the presence of a characteristicenzyme present in the sample. By detecting an enzyme associated with aparticular phenotype of the microorganism, the presence of a bacterialphenotype having a particular function can be detected.

A preferred method of identifying organisms (or biomarkers associatedwith organisms) is mass spectrometry (MS). Certain mass spectrometrictechniques are highly sensitive and are capable of detecting smallquantities of analyte, even in the presence of other potentialinterfering materials. One particularly preferred MS is matrix-assistedlaser desorption/ionization time-of-flight mass spectrometry (MALDI TOFMS). MS has been used to identify organisms using intact cells or spores(see, e.g., Krishnamurthy and Ross, Rapid Commun. Mass Spectrom.10:1992-1996 (1996); Leenders, F. et al. Rapid Commun. Mass Spectrom.13:943-949 (1999)). MS analysis of cellular constituents such asproteins, nucleic acids, and lipids has also been reported (see, e.g.,Demirev, P. A. et al., Anal. Chem. 71:2732-2738 (1999)). The use ofprotein database searching has increased the speed and versatility ofthe analysis when proteins are used to identify the organism (see, e.g,Perkins, D. N. et al., Electrophoresis 20:3551-67 (1999)). For example,the NCBI and Mascot databases contain theoretical peak data that can beused to identify proteins. Data from mass spectrometric analysis can bemanipulated and analyzed using commercially-available software such asData Explorer (Applied Biosystems).

Protein sequencing of enzymatic digests using multidimensional MStechniques (MS^(n)) including tandem mass spectrometry (MS/MS)) can alsobe used to identify organisms or expressed gene products in themicrocosm of the invention. Such proteomic approaches permit rapid,highly automated analysis (see, e.g., K. Gevaert and J. Vandekerckhove,Electrophoresis 21:1145-1154 (2000)).

The identification of microorganisms by MS can advantageously beperformed by isolation of the microorganism, followed by MS analysis ofeither the whole cell, or proteins, such as the ribosomal proteins.Robotic devices can be integrated with MS instruments to provide forautomation of this analysis technique. For example, commerciallyavailable robotics allow for fully automated sample preparation andanalysis, including sample clean-up and concentration, preparation andimaging of two-dimensional (2D) electrophoresis gels, harvesting andenzymatic digestion of the protein spots, and preparation of theenzymatic digests for MS analysis. One commercially-available system forhigh-throughput in-gel digestion, sample cleanup, and MALDI spotting isthe MultiPROBE II Proteomics Workstation from PerkinElmer, Inc. Analternative to gel electrophoresis for protein isolation is liquidchromatography (LC); LC-MS (including LC-MALDI TOF) interfaces have beenreported (see, e.g., U.S. Pat. No. 6,140,639).

However, MS analysis can be complicated when more than one microbialspecies is present. One way of simplifying the analysis is by use ofisotopically-labeled substrates for microbial growth. Thus, if¹³C-labeled carbon sources are provided, an organism capable ofmetabolizing that carbon source will incorporate ¹³C if the organismgrows under the ambient conditions in the ISMA. MS detection andisotopic comparison with reference materials can reveal which biomarkershave incorporated ¹³C; in the case of mixed cultures of microorganisms,these biomarkers can also be identified to determine whichmicroorganisms were capable of growth using the isotopically-labeledcompound as a carbon source in situ. Similarly, isotopically labeledmicroorganisms can be tested in the ISMA to determine their behavior inan environment of interest and to study the flow of energy in a complexsystem via tracing of the isotopes.

The present inventor has found that identification of certain proteinscharacteristic of a specific organism can be achieved by massspectrometric (e.g., MALDI TOF MS) analysis of cell lysates. Forexample, Sphingomonas wittichii Strain RW1 is a bacterium capable ofmineralizing dioxin and related compounds (see, e.g., Halden, R. U,“Engineered in situ biodegradation of dioxins and related compounds”Ph.D. Thesis. University of Minnesota, Minneapolis, Minn. (1997)). Thebiotransformation activity of this organism is due, at least in part, tothe presence and concentration of dioxin dioxygenase, an enzyme whichinitiates degradation of dioxins and dibenzofuran. The presence of S.wittichii in culture can be determined by MALDI TOF detection of dioxindioxygenase in tryptic digests of RWI cells. While detection of dioxindioxygenase in whole cells appeared possible, better results wereobtained through sonication of whole cells, preferably followed bycentrifugation of the whole cell extract and analysis of the supernatant(see, e.g., FIG. 6 for a description of the analysis). Under theseconditions, the presence of the alpha subunit of dioxin dioxygenase, andtherefore the presence of S. wittichii in culture, can be determinedwhen at least 10⁶-10⁷ cells (grown in the presence of dibenzofuran) wereanalyzed by MALDI TOF MS with protein database searching.

Parasites can also be detected in an analogous fashion. Thus, forexample, Schistosoma mansoni, a parasite transmitted through water, wasdetected by isolation of parasitic cercariae (larval stage), sonicationof whole cercariae, centrifugation, trypsin digestion of the supernatant(containing soluble proteins), and MALDI TOF MS analysis. Comparison ofMS-detected peptide fragments to the NCBI metazoan database yielded abest match to a stathmin-like protein of S. mansoni using peptide massfingerprinting in conjunction with one-dimensional MS. This method israpid and specific for the detected parasite, and provides informationregarding the presence or absence of infectious forms of the parasite,by detecting the stathmin-like protein that is expressed only ininfectious lifecycle stages of the parasite. This method provides arapid method for sampling and testing water sources to determine whetherinfectious schistosomal parasites are present. In addition, the devicesof the invention can be used in situ to explore conditions suitable foreradication of such parasites. The confidence of identification of suchdetections can be raised by further analyzing detected characteristicmasses using multi-dimensional MS.

In addition to, or as an alternative to, MALDI TOF MS analysis,multidimensional mass spectrometric analysis (e.g., MS/MS) can be usedto identify proteins. If the number of target peptides is insufficientfor successful detection by peptide mass fingerprinting (PMF),fragmentation of detected biomarkers and MS/MS of the resultant peptidefragments can be used to sequence the peptides and proteins of interestaccording to well-known methods. Peptide sequencing by MS/MS and MS^(n)can be used alone, or as a confirmation of a protein identified bydatabase matching in a MALDI TOF analysis. Similarly, undigested targetbiomarkers can be detected by MALDI TOF MS in linear mode and theiridentity confirmed by MS/MS analysis of fragments produced during tandemmass spectrometry according to well-known methods.

Another useful technique for identifying microorganisms is the use ofarrays of nucleic acid probes on a “chip” (see, e.g., A. Troesch et al.,J. Clin. Microbiology (1999) 37(1): 49-55). In this method, nucleicacids from a microorganism are isolated, amplified if necessary (e.g.,by polymerase chain reaction (PCR)), and selective binding to an arrayof nucleic acid probes is used to identify the microorganism. Theisolation and amplification steps are preferably performed in anautomated fashion using commercially-available robotic equipment.

Other methods for identification of cells, proteins, or other compoundsof interest can be used. For example, cells (including bacteria), viralparticles, or antigenic proteins can be identified by standardimmunoassays, using an antibody to the material of interest. Theantibody can be labeled in any conventional fashion, and binding of theantibody (indicating the presence of the target) can be detected by avariety of methods (detection of radioisotopes in radioimmunoassay,colorimetric detection for enzyme-linked immunosorbent assay (ELISA),chemiluminescent detection, and the like). Other protein identificationmethods include 1- and 2-dimensional electrophoresis, immunoblotting,affinity chromatography, and other standard techniques.

Systems

In another aspect, the invention provides a system for characterizingmicroorganisms present in an environment. The system includes a) acollection device having a plurality of capillary microcosms forcollecting microorganisms or test compounds; b) a sampling device forsampling or manipulating microorganisms present in the capillarymicrocosms; and optionally c) an analysis device, such as a massspectrometer, for characterizing the microorganisms in the capillarymicrocosms; wherein the sampling device is adapted to provide aplurality of samples to an analysis device.

The collection device of the system is a device as described hereinabovehaving an array of capillary microcosms. As described above, inpreferred embodiments the capillary array contains a standardized numberof capillary chambers (e.g., 96, 384, 1536) corresponding in size andconfiguration to standard microtiter plates. By using a standard sizeand footprint for the capillary array, it is possible to usestandardized robotic handling equipment (e.g., liquid handlers) to load,unload, or analyze the contents of the chambers.

The system includes a sampling device for sampling microorganisms ortest compounds present, in the capillary arrays, e.g., after removal ofthe collection device from the test environment. The sampling device ispreferably an automated sample handler, such as an automated liquidhandler or other sampling handling station. Commercial liquid handlerscompatible with standard 96-well microtiter plates are readilyavailable.

The system also includes an analysis device such as a mass spectrometerfor characterizing the microorganisms or test compounds present in thecapillary microcosms. Certain mass spectrometric methods forcharacterizing microorganisms or test compounds have been describedabove, and others are known in the art. Preferred mass spectrometrictechniques are MALDI TOF MS and MALDI MS/MS, atmospheric pressureionization techniques (API) such as electrospray ionization (PSI) MS,ESI-MS^(n) and atmospheric pressure chemical ionization (APCI) MS andAPCI MS^(n); accordingly, in preferred embodiments, the massspectrometer is capable of operating in single MS or multidimensional MSmodes. A variety of commercially available mass spectrometers can beused as the mass spectrometer of the system.

In preferred embodiments, the system includes additional components. Forexample, the sample handler preferably transfers samples from thecapillary array to an automated sample clean-up and preparation station.For example, robotic workstations can provide sample clean-up byfiltration, precipitation, solid-phase extraction, or other knowntechniques for removing extraneous materials and contaminants and/orconcentrating the sample prior to analysis.

Another preferred component of the systems of the invention is anautomated station for performing biomarker purification or separation.In one embodiment, gel electrophoresis (preferably 2D electrophoresis orcapillary gel electrophoresis) is use for biomarker (e.g., protein)purification. Commercially available automated stations for performingcapillary electrophoresis are available (e.g., the ProteomeLab PA 800from Beckman Coulter, Inc.).

In certain embodiments, the system further includes an automatedworkstation for further processing of biomarkers after purification orseparation. For example, digestion of proteins isolated from a gelelectrophoresis separation is used to prepare a sample for MS analysis.As previously described, automated workstations for in-gel digestion,sample cleanup, and MALDI spotting are commercially available.

The components of the system are preferably controlled by a computercontrol and data collection system capable of tracking each sample fromstart (capillary microcosm) to finish (mass spectrometric analysis). Thecomputer system can monitor the automated processes and collect datasuch as retention times (in chromatography or electrophoresis) andmolecular weight of ions in the mass spectrometer. In preferredembodiments, the computer system further comprises a molecular weightfragment database for determining the structure of compounds, or theidentity of peptide sequences, present in the samples. Such systems arereadily available and can be selected according to the type of analyteto be determined.

FIG. 1 shows one embodiment of a system according to the invention.Collection device 100 includes a capillary array 110 of individualmicrocosms 112. Sample handler 120, a sample handling station (e.g., aliquid handling station), removes a portion or aliquot of material frommicrocosms 112, and optional clean-up station 130 performs a sampleprocessing procedure, e.g., a filtration and concentration procedure onthe samples to remove contaminants and concentrate the biomarker ofinterest, if present. Each sample is optionally subjected to biomarkerpurification (e.g., protein purification or separation by gelelectrophoresis) at workstation 140, followed by optional biomarkerprocessing for analysis (e.g., after gel electrophoresis of peptides orproteins, in-gel digestion, sample cleanup, and MALDI spotting) atworkstation 150. Finally, prepared samples are analyzed in massspectrometer 160 (which can be a MALDI mass spectrometer).

The operations are controlled and coordinated by computer 170, and theresults of these operations (data) are stored in central database 180for analysis, including comparison and matching of mass spectral data todatabases, and/or sequencing of peptides from MS^(n) peptide fragmentdata. It will be appreciated that a central computer as shown in FIG. 1is convenient but not required for sample analysis; each individualworkstation can optionally be controlled by an associated computer.

Methods of Diagnosing, Screening and Optimizing Therapeutic Regimens

Another application of the devices and methods of the invention is inthe study or optimization of treatment regimes. It is generallyrecognized that different patients can react in differing ways tostandardized therapies (see, e.g., Mancinelli L, Cronin M, Sadee W.,AAPS PharmSci. (2000) 2(1):E4). For example, genetic differences inliver enzyme profiles can result widely-differing rates of metabolism ofdrugs, resulting in a patient receiving a dose that may be either solarge as to be harmful or so small as to be ineffective. According tothe invention, a variety of therapeutic conditions can be screened insitu, without exposing the patient to any potentially dangerousconditions. Thus, the invention provides personalized, improved methodsfor optimizing therapy and improving patient safety and quality of life.

In one embodiment, the invention provides a method for screeningtreatment conditions without exposing a patient to potential harmfulconditions. In one embodiment, the method includes a) providing a devicecomprising a housing and an array of test chambers, each of the testchambers being in fluid communication with a fluid manifold, and each ofthe test chambers containing either i) test cells or ii) control; b)exposing at least one of the test chambers to a test fluid and acandidate pharmaceutical agent; c) determining the effect of thecandidate pharmaceutical agent on the test cells; and d) selecting anoptimized medical treatment for the patient. In certain embodiments, themethod comprises exposing test cells in a capillary array to a pluralityof treatment conditions, and determining the effect of the treatmentconditions on the cells.

In this embodiment, the capillary chambers of the device will typicallybe charged or loaded with a test sample, e.g., a tissue sample or cellculture of normal or abnormal cells (or both), a sample ofdisease-causing bacteria, or the like. For example, to treat a patientafflicted with cancer, a biopsy of the cancer can be taken by anyconventional method (e.g., needle biopsy), and a sample of the cancerouscells placed in one or more capillary chambers of the ISMA device. Incertain embodiments, a sample of one or more normal cell samples orcultures (e.g., epithelial cells, which are known to be sensitive tomany conventional chemotherapeutic agents), either from the patient or anormal control, will also be present in one or more of the capillarychambers. The microcosm chambers can be provided with any media requiredfor maintaining the viability of the tissue sample, such as growthmedia, growth factors, and the like.

When the chambers have been prepared with test samples, the device canthen be contacted with or immersed in a test fluid, such as a biologicalfluid (such as whole blood, serum, or plasma) drawn from the patient.Alternatively, the device can be connected to a fluid conduit, such asan intravenous line or a catheter, to provide a bodily fluid to thedevice (which is located ex vivo). In still another embodiment, thedevice is implanted in the patient's body and is in contact with abiological fluid in situ. In embodiments in which blood is used as thetest fluid, the device of the invention preferably is constructed sothat blood-contacting surfaces are made of, or coated with, materialsthat do not cause blood coagulation. Anti-coagulant materials are knownin the art, and include heparin and other compounds.

In certain embodiments, the device can be configured to be wearable onor adjacent to a body surface, e.g., by the patient. In this embodiment,a flexible housing can be used to permit the device to conform to thebody of the wearer. The device can be insulated and/or heated tomaintain a temperature similar to the body temperature of the wearer, inorder to maintain an environment for tissue samples closely simulatingthe in vivo environment.

The effect of different treatment (e.g., chemotherapeutic) regimes(e.g., different agents, time courses, or concentrations ofchemotherapeutic agents) can then be assayed by adding the appropriateagents to the test fluid or fluid stream and observing any effects onthe sample cells. The amount of therapeutic agent, the duration oftreatment, or the combination of different therapeutic agents can beassessed in a single ISMA by directing fluid to the appropriate chambersas the therapeutic compounds are added to the fluid. Alternatively, thechemotherapeutic agents can be provided in the capillary chamberstogether with the cell samples, and the effects on the cells in eachcapillary chamber can be observed independently.

As an example, the effects of chemotherapy on a normal cell culture canbe compared to the effects of the same therapy on a cancer cell biopsysample, to determine whether acceptable anti-cancer effects can beobserved on cancer cells while sparing normal cells. The comparison canbe performed, e.g., by removing the cells from the device and assessingcell viability for normal and cancerous cells, by any of a variety ofwell-known methods. Cells can be assessed to determine, e.g., growthrates, cell cycle progression, expression of gene products related toprogrammed cell death, and the like. In certain embodiments, proteomicanalysis of cells from the microcosms (e.g., normal or cancer cellsafter treatment in one or more microcosms with a potential therapeuticagent) can be performed using MS-based analysis systems as describedabove, e.g., to determine levels of protein expression in normal andcancerous cells before and after treatment to determine which proteinsare affected by the treatment.

In another embodiment, the invention provides a method for optimizingmedical treatment for a patient, in which the method includes the stepsof a) providing a device comprising a housing and an array of testchambers, each of the test chambers being in fluid communication with afluid manifold, and each of the test chambers containing either i) acandidate pharmaceutical agent or ii) a control; b) exposing each of thetest chambers to a biological fluid of the patient; c) determining theeffect of the candidate pharmaceutical agent on the biological fluid;and d) selecting an optimized medical treatment for the patient.

It will be appreciated by the skilled artisan that in certainembodiments, the device of the invention can be used to determine theeffects of potential treatment regimens without exposing the patient tothe treatment itself. Because the test compounds and effluent from thetest chambers can be collected and retained within the housing, thepatient is never exposed to any potentially harmful materials. Thisfeature of the invention permits the collection of data on human oranimal response to chemicals, materials or conditions that cannot easilybe studied by conventional means (e.g., for ethical reasons).

For example, potential drugs contained within the test chambers of thedevice can be exposed to a biological fluid of the patient, such asblood, which is admitted into the fluid manifold of the device. Eachchamber can contain, e.g., a control (such as an inert polymer matrix,or a sample of normal tissue or cells) or a candidate pharmaceuticalagent or mixture of agents. In chambers containing a candidatepharmaceutical agent, following exposure of the biological fluid to thecandidate agents in the test chambers, the effect of the potentialtreatments can be determined. As described above, the devices andmethods of the invention provide a convenient and ethically-acceptablemeans for performing research on human or animal interactions with anytype of material or life form.

In certain embodiments, the candidate pharmaceutical agent comprises,e.g., an antibiotic, an antineoplastic agent, an antidiabetic agent, ananticoagulant agent, an anti-inflammatory agent, a vaccine, ananti-angiogenic agent, or a natural or synthetic nucleotide,polynucleotide or polynucleotide mimetic (such as DNA, RNA, or peptidenucleic acid (PNA)). In certain embodiments, the biological fluid isblood. In certain embodiments, the device can be miniaturized andadapted for implantation into the patient's body, while in otherembodiments, the device can be adapted for connection to a fluid conduitsuch as an intravenous line or catheter.

In certain embodiments, the step of determining the effect of thecandidate pharmaceutical agent on the biological fluid comprisesdetermining the bioavailability, biodistribution, biostability ormetabolism of the candidate pharmaceutical agent in the biologicalfluid. For example, after exposure of blood to a candidatepharmaceutical agent in the device, the concentration of the drug can bedetermined, either through sensors embedded in the device, or byremoving samples from the test chambers for laboratory testing. Theconcentration of drug in blood cells, e.g., white blood cells, or theamount of drug bound to circulating protein, can be determined byseparating these components from whole blood and measuring drugconcentrations. Similarly, the ability of a candidate drug to penetratecell layers (e.g., in a tissue biopsy sample) can be readily determinedusing the devices and methods described herein. Such determinations canbe used to predict whether a treatment will be effective when the drugis dosed to the patient, without the need for actually administering thedrug to the patient. Different amounts of drug, formulations of drug(e.g., extended release formulations), and other parameters can besimilar tested in a rapid and safe fashion.

In certain embodiments, the step of determining the effect of acandidate pharmaceutical agent on the biological fluid (or test cells)comprises determining the antibiotic effect of a candidatepharmaceutical agent on a blood-borne pathogen. For example, in certainembodiments, at least some of the test chambers can include filtersand/or growth media for culturing microorganisms. For example, if thecandidate suffers from an infection, the device can be used to determinethe type and strain of the microorganism responsible for the infectionin much the same way that other environmental microorganisms can bestudied with the devices of the present invention, as described above.Still other test chambers can include both a filter and/or growth mediumfor culturing microorganisms and an agent for treating a diseasecondition caused by the microorganism, for example, an antibiotic. Theinvention thus provides methods for simultaneously determining whichmicroorganisms are present in a biological fluid, and which treatmentsare likely to be effective in treating the infection.

In a related embodiment, the invention provides devices and methods fordetermining whether a subject suffers from parasitic infection, andoptionally for determining an effective treatment for such parasiticinfection. Exemplary parasites include Schistosoma mansoni, Plasmodiumfalciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malaria,Leishmania donovani, Treponema pallidus, and the like. These parasitescan be trapped or captured by placement of a device of the invention ina biological fluid (such as blood, urine, or feces) and test compoundspresent in certain of the chambers of the capillary array (or added fromreagent reservoirs) can be screened to determine an effective compoundand dosage level.

In another embodiment, the step of determining the effect of thecandidate pharmaceutical agent on the biological fluid comprisesdetermining the ability of a candidate pharmaceutical agent to stimulateproduction of cell signals such as cytokines, hormones, nitric oxide,and other intercellular or intracellular signaling molecules.

Example 1

One embodiment of the present invention takes the form of an in situmicrocosm array (ISMA) sampler or testing device 1. This embodiment ofthe device is well-suited for analysis of environments such assub-surface water sources or aquifers. As shown in FIGS. 1-4, itsprincipal components include: a housing or container 10 having a fluidinlet 12 and outlet 14, a plurality of capillary microcosms 16 situatedwithin this housing, with these capillaries 16 making up what isreferred to as a microcosm array, each of these capillaries 16 having aninlet 18 and outlet 20 that are configured so as to allow for fluid flowthrough the capillaries 16, each of these capillaries contains anoptional filtration material 22 that is selected for its ability tofoster microorganism collection in the individual capillaries, upper 24and lower 26 valve plates having openings 28 that are configured to bealignable with the capillary inlets 18 and outlets 20, a pneumaticcylinder 30 with coupling means 32 and an assortment of springs 34serves to enable these valves to be moved laterally so as to open orclose the capillaries' inlets 18 and outlets 20, gasketing pads 36, 38serve to prevent leakage from these openings, a pump 40 is connected tothe container's inlet 14 and is sized so that it can draw fluid from theenvironment surrounding the container 16 and push it through thecontainer's inlet 12 and through the capillaries 16, a collecting deviceor a bladder 42 is connected to the pump's outlet and is used to collectthe flow through the container 16, a check valve 44 connected betweenthe pump 40 and bladder 42 prevents backflow of fluid through thecontainer 16, a weight 46 serves to provide ballast for suspending viaan umbilical cable 48 the sampler 1 down a suitably drilled well thatextends into a region of interest.

In this Example, the dimensions of the device were based on commerciallyavailable 96-position (8 wells by 12 wells) microtiter plate format(e.g., Wheaton Scientific Products); similar 384, 1536 (or more) plateformats can also be used. Each well or “microenvironment” of the deviceconsisted of a Teflon block with 96 drill holes representing individualmicrocosm capillaries (1.12 mL; 0.295 inch in diameter by 1 inch inlength).

The inclusion into the ISMA sampler 1 of a pump 40, closure mechanismvalve plates 24, 26, and semi-permeable membranes allows one to firstinoculate and then incubate the device in the environment withoutremoving (and potentially harming) the resident microbes from theirnatural environment. Pump configurations other than those shown in thedrawings include, but are not limited to, multi-channel pumps and pumparrays that deliver fluids to the inlet of one or more individualmicrocosm capillaries.

The ISMA sampler 1 of the present invention can be equipped with acollecting device or a bladder 42, which in this Example is locatedoutside the housing 10 for the capillary array. Fluid flowing throughthe array exits the container through a fluid-tight connection and iscollected in the bladder. Displacement of air from the collection devicemay be desirable, and can be achieved by inclusion in the collectiondevice of a bleed valve allowing air to escape via a piece of tubingrising along the umbilical cable to a location some distance above thefluid intake.

As the fluid from the environment flows through the device,microorganisms and chemicals can be trapped in the capillary microcosms16. When the collection device is full, a float or other fluid sensorcan trip power to the pump and actuate the valve plates 24, 26 of theclosure mechanism, thereby sealing the array. Immediately, or after anadditional incubation period in batch mode, the device 1 can be removedfrom the environment for further analysis.

Example 2

In this Example, cells of a bacterium (S. wittichii strain RW1) wereidentified using MALDI TOF mass spectrometry and multidimensional massspectrometry in conjunction with peptide mass fingerprinting and peptidesequencing.

Culturing of Strain RW1.

Liquid cultures of S. wittichii strain RW1 (DSMZ 6014) were grown at 30°C. in a water bath shaker in M9 phosphate-buffered minimal mediumsupplemented with (i) dibenzofuran (DF) crystals (Sigma-Aldrich;Milwaukee, Wis.), (ii) 50 mM glucose, or (iii) both. Saturated DF mediumcontained approximately 3-5 mg l⁻¹ of the binuclear aromatic compound inthe dissolved phase. Turbidity of the cultures was monitored using aDR/4000U spectrophotometer (Hach, Loveland, Colo.) at a wavelength of560 nm. Viable bacteria were enumerated by plate counts using M9 mediumsupplemented with 1.5% agar (Difco, Franklin Lakes, N.J.) and 5 mMsodium benzoate. Negative control samples composed of cells of RW1lacking the dioxin dioxygenase were obtained via growth of the bacteriumon non-selective Luria Bertani broth, a complex medium that repressesdioxygenase expression.

Microorganisms Serving as Negative Controls.

More than 20 different Proteobacteria served as negative controlsthroughout this study. Most of these represented poorly characterizedenvironmental monocultures and mixed cultures that had been obtained viaselective enrichment using dioxin-like compounds as sole sources ofcarbon and energy. Pseudomonas putida KT2440 (DSMZ 6125) was the onlynegative control strain for which the complete genome was available insearchable online databases. All cultures were grown in selectiveconditions on aromatic substrates to maximize the expression ofaromatic-ring dioxygenases.

Sample Preparation.

Four different types of cell preparations were furnished for MALDI-TOFMS. Cells growing in the early, mid and late exponential phase wereharvested by centrifugation. (3,000×g, 30 minutes, 4° C.), washed, andresuspended in 50 mM NH₄HCO₃ (Fraction 1; undisrupted cells). Biomasswas disrupted on ice using a Sonic Dismembrator (Fisher Scientific,Pittsburgh, Pa.) on low setting for three bursts of 10 s, with coolingperiods of 30 s between bursts, yielding Fraction 2 (disrupted cells).Sonicated cell suspensions were centrifuged (13,500×g, 5 minutes, 4° C.)to separate the supernatant of the crude cell extract (Fraction 3; wholecell extract) from the pellet (Fraction 4) consisting primarily of celldebris and undisrupted whole cells. For experiments involving2-dimensional gel electrophoresis, whole cell extracts were divided intotwo equal volumes (sample splits) to allow for additional analysis byMALDI-TOF MS; reported CFU in the sample are corrected for the loss ofbiomass resulting from splitting of the samples.

In Silico Digestion.

Peptides resulting from tryptic digestion of the alpha- andbeta-subunits of the dioxin dioxygenase were predicted from sequencesdeposited in the NCBI database (http://www.ncbi.nih.gov/) usingMS-Digest (http://prospector.ucsf.edu/). Screening of combinations ofvarious search parameter settings resulted in the following optimalsettings: the in silico digests were performed using trypsin anddisallowing missed cleavages or post-translational modifications.Cysteines were presumed to be unmodified; as were the N- and C-terminiof the peptides. The mass range was specified as 500-5,000 Da; multiplycharged ions were not considered.

MALDI-TOF MS Analysis.

Samples (25 ul) were digested with 200 ng trypsin in 50 mM NH₄HCO₃ at37° C. overnight, vacuum-dried in a Savant SVC100 Speed Vac (GMI,Albertville, Minn.), desalted using C₁₈ Omix microextraction column tips(Varian, Palo Alto, Calif.) and mixed with matrix solution (˜1.5 ul)consisting of 10 mg ml⁻¹ of alpha-cyano-4-hydroxy-cinnamic acid (CHCA)in 50% acetonitrile and 0.1% trifluoroacetic acid (TFA). A stainlesssteel 96-well MALDI target plate (Applied Biosystems, Foster City,Calif.) was spotted with approximately 1 ul of the sample/matrixsolution, which was then air-dried. Spectra were acquired using aVoyager DE-STR MALDI-TOF MS (Applied Biosystems, Foster City, Calif.) inpositive reflector mode (m/z 500-5000; 50 laser shots per spectrum).Initial external calibration was performed using a standard peptidemixture (human bradykinin fragment 1-7, 757.3997 Da; humanadrenocorticotropic hormone fragment 18-39, 2465.1989 Da; bovine insulinchain B, oxidized, 3494.6513 Da) purchased from Sigma (St. Louis, Mo.).Additional internal calibration was carried out as described below,

Mass Spectral Data Analysis.

Mass spectral data were analyzed and manipulated using Data Explorersoftware (Applied Biosystems, Foster City, Calif.). Spectra weredeisotoped using the manufacturer's settings. Internal calibration wascarried out using trypsin autolysis peaks. Acquired data were analyzedby comparison to in silico information contained in the NCBI databases(http://www.ncbi.nih.gov) using PMF. The 300 most intense peaks weresearched against the NCBI taxonomy subset “All Bacteria” (753,000+sequences) at a mass tolerance of 50-100 ppm using MASCOT. Additionalsearch parameters included disallowing for missed cleavages and eitherfixed or variable post-translational modifications. Probability scoresfor positive identification were determined using the statisticalalgorithm in the program described elsewhere for peptide-massfingerprinting (PMF) (Pappin, D. J. C., P. Hojrup, and A. J. Bleasby,Curr. Biol. 3:327-332 (1993).

Peptide Sequencing.

Protein identifications obtained by PMF were confirmed in selectedsamples via sequencing of the target mass at m/z 3036.3 using an iontrap mass spectrometer (LCQ Deca XP; Thermo Electron Corporation, MA) inconjunction with an atmospheric pressure MALDI source (Mass Tech Inc.,MD). Presence of the alpha-subunit of the dioxin dioxygenase wasconfirmed by submission of detected fragment ions to the Sequestdatabase.

Results

In Silico Analyses.

Theoretical (in silico) digestions were performed to construct peptidemaps of the large (alpha-) and small (beta-) subunits of the dioxindioxygenase. The porcine protease used in this study, trypsin(E.C.3.4.21.4), cleaves proteins after the amino acids lysine andarginine, unless these are followed by a proline. Digestion yieldedindividual amino acids and peptides, the latter ranging in length from2-35 amino acids. For the alpha-subunit, there were 31 predictedpotential MS targets in the experimentally defined detection range(mass-to-charge ratios of m/z 500-5,000), covering 94% of the aminoacids of the total protein. The remaining 6% of the protein mass wascomposed of peptides situated outside of the detectable range (m/z<500). The beta-subunit was calculated to yield a maximum of 15detectable tryptic peptides, suggesting a maximum theoretical proteincoverage of 84% (data not shown).

Screening of Various Fractions of RW1 Cells for the Dioxin Dioxygenase.

Initial experiments concentrated on the feasibility of detecting thedioxin dioxygenase in four different fractions of processed cellcultures (see FIG. 6). Proteins contained in the various cell fractionswere digested, purified and desalted via passage through a pipet tipfunctioning as a C₁₈-microextraction column. Purified digests were mixedwith matrix, and analyzed by PMF using MALDI-TOF MS as shown in theschematic (FIG. 6). Investigated cell fractions included undisruptedcells (Fraction 1), cells disrupted by sonication (Fraction 2), wholecell extracts representing the supernatant of disrupted, centrifugedcells (Fraction 3), and the corresponding pellet consisting of celldebris and residual whole cells (Fraction 4). Since the optimal amountof biomass for the assay was not known a priori, experiments wereperformed using a range of initial cell quantities (10⁵-10⁸ cells).

Fraction 1.

Analysis by peptide mass fingerprinting of a digest of 10⁸ undisruptedcells of RW1 yielded ten target peaks above the baseline noise: m/z685.4, 951.5, 1,234.6, 1,393.7, 1,541.8, 1,847.8, 2,005.0, 2,194.0,2,222.1, and 3,036.3. A list of 300 ions having the greatest signalintensities was generated and submitted to online protein databasesrepresenting the kingdom of Bacteria. The data query returned thealpha-subunit of the dioxin dioxygenase as the best fit among 753,000+proteins. The resultant Mascot score of 52 indicated that the searchresult was not statistically significant (p>0.1), however. Overall, the10 target peptides provided 31% protein coverage.

Fraction 2.

Analysis of a digest of 10⁷ disrupted cells of RW1 resulted in detectionof nine target peptides of the alpha-subunit of the dioxin dioxygenase.Compared to Fraction 1, the mass at m/z 951.5 was missing and anincrease in the level of noise was observed in the range from m/z1,000-3,200. Again, database searching returned the alpha-subunit of thedioxin dioxygenase as the best match, with a statistically significant(p<0.05) Mascot score of 69. Protein coverage was 32%.

Fraction 3.

Analysis of supernatant obtained by centrifugation of 10⁷ disruptedcells of RW1 yielded the best result. The mass spectrum had a very lowlevel of noise across the entire m/z range of interest. Major detectableions were clustered between m/z 500 and 3,200. In the spectrum shown,four of the eight most intense peaks—detected at m/z 1,393.7 (100%relative intensity), 586.3 (22%), 2,222.1 (17%), and 962.5 (15%)—matchedin silico values calculated for peptides of the alpha-subunit of thedioxin dioxygenase; the second intense ion at m/z 842.5 corresponded toa trypsin autolysis product that was used as an internal standard formass calibration. A total of 13 target peaks were detected, resulting inconfident protein identification (p<0.00001) by Mascot searching, with ascore of 105 and a protein coverage of 34%. Target ions detected atlesser intensities included m/z 685.4 (13% relative intensity), 919.4(7%), 951.5 (13%), 1,234.6 (9%), 1,541.8 (8%), 2,005.0 (10%), 2,194.0(11%) and 3,036.3 (4%). The ease of detection of the alpha-subunit inwhole cell extract is consistent with previous reports that localizeddioxin dioxygenase activity to the soluble proteome of extracts fromcells grown on DF.

Fraction 4.

Analysis of digested pellets obtained by centrifugation of disruptedcells yielded noisy mass spectra that did not show any target m/zregardless of the amount of biomass processed. This finding wasconsistent with literature indicating cell pellets to be depleted indioxin dioxygenase activity relative to whole cell extracts of RW1(Fraction 3). Overall, the results demonstrated that the dioxindioxygenase is most easily detectable by PMF in digested whole cellextract. Therefore, the sensitivity of PMF analysis was furtherinvestigated in the latter matrix.

Sensitivity Analyses and Robustness of the Assay.

To determine the biomass range suitable for positive identification ofStrain RW1 via PMF of the alpha-subunit of the dioxin dioxygenase, wholecell extracts of 10⁵-10¹⁰ DF-grown CFU were analyzed following digestionwith a standard amount of 200 ng of trypsin. Positive proteinidentification with significant probability-based Mascot scores of >68(p<0.05) were obtained consistently when >10⁶ cells were processed andanalyzed. Analysis of extracts obtained from 10⁷ and 10⁸ DF-grown cellsyielded Mascot scores ranging from 73 to 105 (p<0.01-0.00001) and 84 to111 (p<0.001-0.00001), respectively; in these experiments, the number ofmatched peptide masses ranged from 10 to 13 and 12 to 14, respectively,with protein coverages for the alpha-subunit of the dioxin dioxygenaseranging from 31-34% (10⁷ CFU) and from 37-43% (10⁸ CFU). Analysis of<10⁶ CFU yielded no target ions and no significant matches for eitherthe two target proteins or any of the more than 753,000 proteinscontained in the non-redundant NCBI database at the time of dataanalysis. Similarly, no database matches were found in experimentsusing≧10⁹ CFU. A total of 15 different peptide masses, corresponding tothe alpha-subunit of the dioxin dioxygenase, were detected in more than100 experiments conducted with biomass harvested in the early, mid andlate exponential growth phase (total protein coverage of 45%). Incontrast, none of these target peptides were found and no positiveidentifications of the dioxin dioxygenase were obtained during analysisof the more than 20 negative control strains that represented a broadspectrum of microorganisms capable of catabolizing dioxin-relatedaromatic compounds.

Results of repeatedly performed experiments were very consistent. Thefollowing variables had no detectable effect on the outcome of theexperiment (data not shown): substituting alpha-cyano-4-hydroxy-cinnamicacid (CHCA) for 3,5-dihydroxybenzoic acid (DHB) as the ionizationmatrix, type of C₁₈-microextraction column used (n=2), and identity ofthe operator (n=3). However, when cells were harvested late into theexponential growth phase (deceleration phase), a slight drop in Mascotscores was observed.

Interestingly, the beta-subunit of the dioxin dioxygenase was neveridentified by database searching in any of these experiments. This issurprising because the observed removal of DF during growth of RW1cultures indicated the presence of this essential protein at quantitiesequimolar to those of the alpha-subunit. Although some target ions ofthe beta-subunit were present, as determined by manual identification,the signal intensities of these peptide masses at m/z 563.4 (1% relativeintensity), 607.3 (2%), 693.3 (4%), 832.5 (8%), 848.5 (10%), 1,077.6(6%) typically were at or near the baseline noise level. Followingspectral processing and data reduction using a peak threshold ofapproximately 5-10% relative intensity, these ions mostly were rejectedand did not enter into the online database query; this effectivelyprevented a potential identification of the beta-subunit when using theonline search algorithm.

Effect of Growth Substrate on Strain Identification.

Cultures of RW 1, grown in phosphate-buffered mineral salt solutionsupplemented with the growth substrates (A) DF, (B) DF plus glucose, and(C) glucose only, were processed and analyzed by MALDI-TOF MS and 2D gelelectrophoresis. The alpha-subunit of the dioxin dioxygenase—i.e., thepreviously established biomarker of dioxin degradation-enabled cells ofRW1—was identified readily in the digested soluble proteome of DF-growncells, with scores as high as 111, indicating a very low probability offalse-positive misidentification (p<0.00001). Detection of up to 14target peptides in whole cell extracts of 10⁸ CFU resulted in a proteincoverage of 43%, the best result achieved. Again, the ions correspondingto peptides of the alpha-subunit were among the most prominent in themass spectra.

The alpha-subunit of the dioxin dioxygenase also was returned as thebest database match when analyzing glucose-grown cells of RW1 that wereco-exposed to DF for enhanced expression of the dioxin dioxygenase;however, the corresponding score was not significant (p>0.05),necessitating peptide sequencing for unambiguous protein identification.Compared to DF-grown cells, the signal intensity of target peaks waslower in glucose-grown biomass co-exposed to DF. No target peaks weredetected when analyzing biomass grown on glucose in the absence of DF,and no significant matches were found for any of the 753,000+ proteinscontained in the non-redundant NCBI database. Analysis of cells grownusing non-selective complex media, e.g., Luria Bertani broth, alsorevealed no ions of interest in the mass spectra recorded. Lack ofdetection of the alpha-subunit in LB-grown cells was consistent withliterature information indicating repressed dioxin dioxygenaseexpression during growth of RW1 on complex media.

The present study employed PMF on minimally processed microbial cells.Experimental results of the present study revealed the value and powerof this non-traditional usage of PMF by MALDI-TOF MS for theidentification and phenotypic characterization of certain environmentalmicroorganisms such as Strain RW1.

In contrast to mass spectral microbial fingerprinting, PMF is morepowerful because specific target proteins can be selected a priori andtheir corresponding ions (peptide masses) can be predicted in silico.Identification is based on the detection of multiple fragments of agiven protein rather than on a single molecular ion. Therefore, proteinmatches by PMF have a quantifiable confidence level and often arestatistically highly significant even when searching non-restricted,complex databases containing hundreds of thousands of proteins. Theidentity of detected proteins can be ascertained without having toobtain and analyze authentic protein standards, an important advantagewhen attempting to identify environmental isolates whose proteins havenever been purified. Since the function of the detected biomarker eitheris known or can be inferred, PMF of microbial cells can reveal criticalinformation on biomass physiology that otherwise would be difficult orimpossible to obtain.

The analysis strategy and methodology presented in this Example isattractive for application, e.g., in the field of bioremediation forseveral reasons. One advantage of this assay is its ability to identifycells of RW 1 and simultaneously yield information on their mostcritical phenotypic characteristic that drives the removal of dioxinsfrom contaminated environments during bioaugmentation: the expression ofappreciable quantities of the dioxin dioxygenase. Analysis of whole cellextracts by PMF can inform on the extent to which vegetative cells ofRW1 are charged with this enzyme. Since the assay is performed on anon-purified bacterial proteome fraction, only cells containingappreciable quantities of the dioxin dioxygenase are detectable by PMF.

The methodology demonstrated here for a dioxin-degrading bacterium maybe extended to other microorganisms containing large quantities ofcharacteristic proteins. Enzymes expressed at moderate quantities alsomay be suitable targets as long as their corresponding peptides ionizefavorably, similar to those of the alpha-subunit of the dioxindioxygenase. Since the technique is inexpensive and potentially may beautomated, it could prove valuable in bioremediation and other areas ofapplied and environmental microbiology.

Additional benefits of the assay are its reproducibility, robustness andthe potential for unattended high-throughput analysis during routinescreening of environmental isolates.

Example 3

In this Example, a parasite is detected by use of MALDI TOF MS.

Schistosomes are parasites in a wide variety of warm-blooded hosts. Inmammals and humans, they cause serious diseases. It is estimated thathundreds of millions of people worldwide are infected with schistosomes.Infection occurs when cercaria, a free-swimming larval form of theparasite found in contaminated water, penetrate the skin of themammalian host.

Detection of the cercariae in water is conventionally performed byskimming the cercariae from the surface of the water, followed by visualobservation by a parasitologist. As an alternative, use of MALDI TOF MSwas investigated. A schematic representation of the detection method isshown in FIG. 7 (some optional steps are also shown in FIG. 7).

Methods.

Lyophilized cercariae of Schistosoma mansoni were divided into fractionsof approximately 2,500 cercariae and resuspended in 50 mM ammoniumbicarbonate. Cells were disrupted using a Fisher 550 Sonic Dismembrator(Fisher Scientific) and spun at high speed (13,500 g, 5 minutes, 4° C.in a Beckman Microfuge 18) to remove cell debris. The supernatant wasassayed colorimetrically for protein content using the bicinchoninicacid assay (Pierce, Rockford, Ill.).

Samples containing 400 ug protein were digested with 200 ng proteomicsgrade trypsin (Sigma, St. Louis, Mo.) in 50 mM ammonium bicarbonatebuffer at 37° C. for 18 hours, then vacuum dried and desalted using C₁₈Omix microextraction column tips (Varian, Palo Alto, Calif.) and mixedwith matrix solution (alpha-cyano-4-hydroxy-cinnamic acid (CHCA) in 50%acetonitrile and 0.1% trifluoroacetic acid (TFA)) prior to deposition ina 96-well MALDI target plate (Applied Biosystems, Foster City, Calif.).Spectra were acquired using a Voyager DE-STR MALDI-TOF MS (AppliedBiosystems, Foster City, Calif.) in positive reflector mode (m/z500-5000; 50 laser shots per spectrum).

Mass spectral data were analyzed and manipulated using Data Explorersoftware (Applied Biosystems, Foster City, Calif.). Spectra weredeisotoped and an internal calibration was carried out using trypsinautolysis peaks. Mass lists were generated and compared to theoreticalpeptides using the NCBI databases and MASCOT.

Results.

Peptide fingerprinting analysis of supernatant from disrupted cells ofcercariae using MALDI-TOF MS and searching of the NCBI metazoan databaseyielded stathmin-like protein gi|3641363 of S. mansoni as the best matchamong over 170,000 sequences searched.

These results show that infectious schistosome cercariae can be detectedsuccessfully using MALDI-TOF MS of minimally-processed crude cellextracts. This novel detection technique shows that automatedhigh-throughput analysis of environmental schistosome parasites isfeasible. The method has sufficient discriminatory power to distinguishbetween schistosomes which infect birds and those which infect humans.This method can be used to detect schistosomes in the environment or inclinical specimens by both one-dimensional and multidimensional massspectrometry.

Example 4

In this Example, a bacterial protein was identified from a complexmixture.

For this Example, a sample mixture was prepared. Samples contained thetarget organism (Sphingomonas wittichii RW1) and four other bacteria (E.colt, Pseudomonas putida KT2440, Enterococcus sp. and Burkholderiaxenovorans LB400). The samples were diluted to 1:1 and 100:1(target:interfering bacteria) and soluble fractions were digested withtrypsin overnight. Known peptides from the target (dioxin dioxygenase)were selected for blind sequencing, and the highest intensity peaks weresubjected to MS/MS and MS^(n) (i.e., multiple-dimension MS, such asMS/MS/MS). Data were analyzed using a Kratos Axima QIT and searchedagainst the NCBInr database using Mascot (www.matrixscience.com).

It was found that the target bacterium (Sphingomonas wittichii RW1) wasnot detected from a mixture of equal amounts of each bacterial type.However, the target bacterial species was successfully identified withconfidence (p=0.00075) from a mixture in which the target representedthe predominant bacterial species (100:1:1:1:1 mixture of bacteriatypes). Also detectable was the dioxygenase gi|37727200 of Pseudomonasputida KT2440.

Example 5

This Example demonstrates the identification of viral protein from acomplex stool matrix.

For this Example, a sample list was created using varying concentrationsof recombinant Norovirus virus-like particles (VLPs) and virus-freestool extracts. Pure VLP stock was used to determine the detection limitof the virus in the MALDI-QIT-TOF. Samples were analyzed by MS, MS/MSand MS³ by selectively targeting the viral peptides. The VLPs werechallenged against increasingly concentrated stool matrices, ultimatelyto a 1× stool concentration. Data were analyzed using a Kratos Axima QITand searched against the NCBInr database using Mascot(www.matrixscience.com).

It was found that the target viral particles could be detected with adetection limit of 10 femtomoles of purified virus particles.

The contents of all patents, patent applications, and publications citedherein are hereby incorporated by reference in their entirety.

Other embodiments are within the following claims.

What is claimed is:
 1. A system for characterizing cells present in anenvironment, the system comprising: a) a collection device having aplurality of capillary microcosms for collecting or maintaining cells inthe environment; b) a sampling device for sampling cells present in thecapillary microcosms; and c) a characterizing device for characterizingthe cells from the capillary microcosms; wherein the sampling device isadapted to provide a plurality of samples to the characterizing device.2. The system of claim 1, wherein the collection device comprises: i) ahousing; ii) an array of capillary microcosms within the housing; andiii) a fluid manifold in fluid communication with the capillarymicrocosms for controllably providing a cell-containing fluid from theenvironment to the capillary microcosms, wherein the housing comprisesan opening to controllably permit cells from the environment to accessthe array.
 3. The system of claim 2, wherein at least one of thecapillary microcosms is provided with a substrate for trapping cells. 4.The system of claim 1, wherein the collection device further comprises apump for pumping fluid into the fluid manifold.
 5. The system of claim1, wherein the collection device comprises at least one sensor forreal-time monitoring of a condition within the collection device.
 6. Thesystem of claim 2, wherein the array of capillary microcosms isconfigured for use with an automated sampling device.
 7. The system ofclaim 6, wherein the sampling device comprises an automated fluidhandling device.
 8. The system of claim 1, wherein the sampling devicefurther comprises means for performing sample clean-up.
 9. The system ofclaim 1, wherein the characterizing device comprises a massspectrometer.
 10. The system of claim 1, wherein the cells are selectedfrom the group consisting of prokaryoyes, eukaryotes, yeasts, fungi,bacteria, Archaea, parasites, protozoa, plant cells, and animal cells.11-22. (canceled)
 23. A method for optimizing medical treatment for apatient, the method comprising: a) providing a device comprising ahousing and an array of capillary microcosms, each of the capillarymicrocosms being in fluid communication with a fluid manifold, and eachof the capillary chambers containing either i) a case sample associatedwith a disease state of the patient or ii) a control sample; b)subjecting each of said case and control samples to a treatmentcondition; c) determining the effect of the treatment condition on eachsample; and d) selecting an optimized medical treatment for the patient.24. The method of claim 23, wherein the treatment condition comprisestreatment with a candidate pharmaceutical agent.
 25. The method of claim23, wherein the control sample is a sample of normal tissue from thepatient.
 26. The method of claim 23, wherein the case sample is a sampleof diseased tissue from the patient.
 27. A method for screening medicaltreatments for a patient, the method comprising: a) providing a devicecomprising a housing and an array of test chambers, each of the testchambers being in fluid communication with a fluid manifold, and each ofthe test chambers containing either i) a candidate pharmaceutical agentor ii) a control; b) exposing each of said test chambers to a biologicalfluid of the patient; and c) determining the effect of the candidatepharmaceutical agent on the biological fluid.
 28. The method of claim27, further comprising: d) selecting an optimized medical treatment forthe patient.
 29. The method of claim 27, wherein the candidatepharmaceutical agent comprises an antibiotic, an antineoplastic agent,an antidiabetic agent, an anticoagulant agent, or a natural or syntheticnucleotide, polynucleotide, nucleotide mimetic, or polynucleotidemimetic.
 30. The method of claim 29, wherein the biological fluid isblood.
 31. The method of claim 27, wherein the device is implantedwithin the body of the patient.
 32. The method of claim 27, wherein thestep of determining the effect of the candidate pharmaceutical agent onthe biological fluid comprises determining the bioavailability,biodistribution, or metabolism of the candidate pharmaceutical agent inthe biological fluid.
 33. The method of claim 27, wherein the step ofdetermining the effect of the candidate pharmaceutical agent on thebiological fluid comprises determining the antibiotic effect of acandidate pharmaceutical agent on a blood-borne pathogen.
 34. A methodfor diagnosing an infectious or parasitic disease condition, the methodcomprising: a) providing a device comprising a housing and an array oftest chambers, each of the test chambers being in fluid communicationwith a fluid manifold and being configured to trap an infectiousmicroorganism or parasite; b) exposing each of said test chambers to abiological fluid of the patient; c) identifying a microorganism orparasite trapped in a test chamber.
 35. The method of claim 34, whereinthe step of identifying comprises identifying a protein characteristicof the microorganism or parasite.
 36. The method of claim 34, whereinthe protein is identified using mass spectrometry.
 37. The method ofclaim 36, wherein the protein is identified using multidimensional massspectrometry.
 38. A method for testing an agent to determine an effectof the agent on a living organism, the method comprising: a) providing adevice comprising a housing and an array of capillary microcosms, eachof the capillary microcosms being in fluid communication with a fluidmanifold, and each of the capillary chambers containing either i) theagent to be tested or ii) a control; b) subjecting each of said agentand control samples to a fluid environment representative of a livingorganism; and c) determining the effect of the agent on the fluidenvironment.
 39. A method for detecting in a sample a microorganismhaving a pre-determined phenotype, the method comprising the steps of:a) selecting a biomarker for the pre-determined phenotype; b) providinga sample for testing; c) detecting by mass spectroscopy the presence orabsence of the biomarker diagnostic of a microorganism having thepre-determined phenotype in the sample.
 40. The method of claim 39,wherein the biomarker is an enzyme.
 41. The method of claim 40, whereinthe enzyme is an oxygenase enzyme.
 42. The method of claim 41, whereinthe oxygenase is a dioxygenase.
 43. The method of claim 39, wherein thestep of detecting by mass spectroscopy comprises detecting an enzyme bypeptide-mass fingerprinting.
 44. The method of claim 43, wherein thestep of detecting is performed on a digest of whole cells.
 45. Themethod of claim 39, wherein the pre-determined phenotype is a phenotypeuseful for bioremediation.
 46. The method of claim 39, wherein themicroorganism is Sphingomonas wittichii Strain RW1.
 47. The method ofclaim 39, wherein the microorganism is a parasite or pathogen.
 48. Themethod of claim 39, comprising the further step of inducing biomarkerproduction in the microorganism before detecting the presence or absenceof the biomarker.
 49. The method of claim 39, wherein the step ofdetecting by mass spectroscopy comprises detecting an enzyme by peptidesequencing using multi-dimensional mass spectrometry.